In recent years, techniques for DNA synthesis have been increasingly developed, for example, in order to construct a long chain DNA molecule having a size of a genome. Known such techniques include assembly of chemically synthesized DNA fragments and PCR-amplified DNA unit fragments. The synthesis process of the chemical synthesized DNA and the PCR method, however, are known to be accompanied by random mutation introduced into synthesized DNA molecules. Therefore, the sequences of the DNA molecules need to be always checked somewhere from the start to the end of the gene-assembling process so as to select a DNA molecule having a desired sequence.
Checking base sequences is usually conducted by Sanger base sequencing on an automated fluorescence sequencer. This method can determine about 800 consecutive bases in a single session of base sequencing. When the number of base sequencing sessions for checking base sequences of chemically- or PCR-synthesized DNA unit fragments prior to gene-assembling is reduced, time and cost can be saved. For this reason, the chemically- or PCR-synthesized DNA unit fragments to be used for gene-assembling are preferably short.
As the DNA unit fragments to be used for gene-assembling become shorter, however, the number of them to be assembled needs to be increased.
A currently known method to assemble a plurality of DNA unit fragments is a gene-assembling method employing a plasmid transformation system in Bacillus subtilis (the OGAB method). Patent Document 1, for example, discloses a method that adopts the OGAB method for constructing a DNA plasmid for use in transforming a Bacillus subtilis cell.
The OGAB method employs a so-called multimeric plasmid, in which multiple plasmid units exist within one molecule by homologous recombination between plasmid molecules. In the OGAB method, the DNA plasmid molecule for transformation is not necessarily circular, but it only has to have a tandem-repeat structure in which a plasmid unit and a DNA unit fragment used for assembling to be assembled appear repeatedly with each unit maintaining a same direction.
In the OGAB method, to prepare a DNA molecule having a tandem-repeat structure as above described, multiple DNA unit fragments, when used, need to be joined to their corresponding plasmids. As the number of kinds of DNA unit fragments increases, it becomes more difficult to join them to their corresponding plasmids and to construct a tandem-repeat structure. To join many DNA unit fragments together, the molar ratio among DNA unit fragments in ligation is desirably made close to 1.
Patent Document 1: Japanese Patent No. 4479199